Molecular mechanisms controlling epigenetic stability of gene expression

In multi-cellular organisms, somatically heritable patterns of gene expression are important for the maintenance of differentiated cell types. The mitotic inheritance of gene expression control occurs, in part, through epigenetic regulation. Genomic imprinting and X chromosome inactivation are examples of epigenetic regulation, which exhibit expression of one allele and silencing of its homologue. The transcriptional activity of these genes can provide an experimental path to measuring the rate and extent of epigenetic stability of gene expression.;An experimental system was developed to determine the role of epigenetic molecular marks of chromatin in the maintenance of transcriptional silencing. Primary mouse fibroblast cultures were established from inter-subspecific F1 female mice carrying a green fluorescent protein (GFP) transgene on the paternal-derived X chromosome. The F1 animals also have frequent DNA polymorphisms between the parental alleles. Allelic RNA transcript ratios were determined at multiple X-linked genes (Utx, Hprt, Pgk1, Xist, Atp7a) and autosomal imprinted loci (H19, Igf2, Snrpn , Mest) using a quantitative primer extension assay. In the same cells, quantitative measurements of allelic DNA methylation were performed for three X-gene promoters. The quantitative relationship between allelic transcription and DNA methylation was determined for the expressed and silenced alleles.;Cells with an inactivated X-linked GFP transgene were treated with epimutagens to determine the frequency of transcriptional reactivation. Significant reactivation of the GFP transgene (∼14%) was found in response to the de-methylating agent, 5-aza-2'-deoxycytidine (5AdC). The treated cells demonstrated a similar, but variable, increase in expression of the silenced allele at the endogenous loci, and a correlation with the expression of the reactivated GFP transgene. Following 5AdC treatment, decreases in DNA methylation were observed within the promoter regions of two X-inactive genes. Specific nucleotide sites exhibited differential methylation that correlated with changes in allelic gene expression. Reactivation of the imprinted genes H19 and Igf2 was independent of methylation changes within the differentially methylated domain (DMD) upstream of the H19 gene. Experimentally induced reactivation failed to support the hypothesis that specific DNA hypermethylation sites are associated with epigenetic transcriptional activity.